Hemoglobin (hereinafter, also referred to as Hb), in particular hemoglobin A1c (hereinafter, also referred to as HbA1c) of a form of glycosylated hemoglobins reflect an average blood sugar level in past 1 to 2 months. Therefore, hemoglobin A1c is widely used in a screening test for diabetes mellitus and as a test item for checking whether a diabetic keeps the blood sugar under control.
Conventionally, HbA1c has been measured by HPLC, immunoassay, electrophoresis or the like. Especially, HPLC is widely used in clinical examinations. HPLC requires only 1 to 2 minutes to measure each sample, and has achieved a measurement accuracy of about 1.0% in terms of a CV value obtained by a within-run reproducibility test. Measurement methods for checking whether a diabetic keeps the blood sugar under control are required to perform at this level.
Meanwhile, application of an electrophoresis technique that enables high accuracy measurement of HbA1c to the clinical examinations is expected to yield a significantly advantageous effect in cost because an electrophoresis apparatus has a simple configuration, and can be formed as a low-cost small system such as a microchip-electrophoresis apparatus.
Measurement of Hb by electrophoresis has been used for a long time to separate abnormal Hbs with an unusual amino acid sequence. However, separation of HbA1c is significantly difficult, and takes 30 minutes or more by gel electrophoresis. Thus, electrophoresis has been unsatisfactory in terms of measurement time and measurement accuracy when applied to the clinical examinations. Therefore, electrophoresis has hardly been applied to clinical diagnosis of diabetes in recent years.
However, capillary electrophoresis, which was proposed in around 1990, generally enables high accuracy measurement with high separation efficiency. For example, Patent Document 1 and Patent Document 2 disclose techniques for separating HbA1c by capillary electrophoresis.
However, use of the method of Patent Document 1 does not overcome the problem of taking a long time to measure, and also may denature Hb due to use of a buffer solution with a high pH of 9 to 12. For these reasons, it has been difficult to apply this method to the clinical examinations.
In the method of Patent Document 2, two techniques that had been known prior to the publication of this Patent Document, that is, a technique using the affinity of sulfated polysaccharides to Hb, and a dynamic coating technique of an inside of a capillary are used in combination. This method enables measurement in a shorter time compared to gel electrophoresis, and takes only about 10 minutes to measure.
However, such a dynamic coating technique requires coating the inside of the capillary after each measurement for the following measurement. Therefore, in order to coat the inside of the capillary in the same way at the time of the beginning of each measurement, it is necessary to remove the coating layer by washing after each measurement to return the inside of the capillary to the initial condition that is the condition before the measurement. Namely, for repetitive measurement, washing and coating procedure needs to be performed between each measurement, resulting in an increase in the measurement time. The washing and coating procedure may also cause a measurement error. In addition, a reagent for coating needs to be prepared for the measurement, leading to a disadvantage in cost. Even when not used for the repetitive measurement, the technique takes about 10 minutes to measure, which is much longer than needed in HPLC, and is unsatisfactory for application to the clinical examinations.
For clinical diagnosis of diabetes, stable HbA1c, which is a type of HbA1c and used as a diabetic indicator, should be separated from substances that disturb measurement such as unstable HbA1c and carbamylated Hbs (hereafter, also referred to as modified Hbs). However, electropherograms obtained by the methods disclosed in Patent Document 1 and Patent Document 2 were unsatisfactory in terms of separation performance and measurement accuracy, and it has been difficult to separate stable HbA1c from the modified Hbs by the techniques within the scope of these methods.
Abnormal Hbs are present in some samples, and known as a diagnostic indicator of a disease due to hemoglobin defect such as hemolytic anemia and thalassemia. Techniques using HPLC for measuring HbA1c have been developed to provide simultaneous measurement of stable HbA1c and the abnormal Hbs. However, such technique using electrophoresis has not been proposed so far.    Patent Document 1: Japanese Kohyo Publication No. Hei-09-510792    Patent Document 2: Japanese Kokai Publication No. Hei-09-105739